Methods: Initially, kinetics, architecture and cellular viability of C. glabrata (ATCC and wild type Cg5) biofilms were evaluated using both traditional (CFU, XTT, spectrometry, SEM) and novel (Confocal- COMSTAT) analytical tools. Then antifungal susceptibility and antioxidant capacities of planktonic vs. biofilm mode of C. glabrata isolates were evaluated using a novel, comprehensive protocol. The protein expression profiles of the planktonic and biofilm mode of C. glabrata were analyzed using two-dimensional difference gel electrophoresis (2D-DIGE) and tandem mass spectrometry (MS/MS). Identity of the proteins and their functional categories were deduced by bioinformatics tools.
Results: C. glabrata biofilms exclusively comprised blastospores embedded in an abundant exopolymeric material. They exhibited 8 to >500 fold resistance to antifungals and higher antioxidative capacities in the biofilm mode compared to their planktonic counterpart. C. glabrata biofilm proteome showed significant upregulation of stress response proteins such as heat shock protein-12 (Hsp12p), cytoplasmic thioredoxin isoenzyme (Trx1p), alkyl hydroperoxide reductase (Ahp1p), vacuolar aspartyl protease (Pep4p) aldehyde dehydrogenases (Ald2p) and alcohol dehydrogenase isoenzyme III (Adh3p). Notably, among the down-regulated proteins, there were several key enzymes involved in the glycolysis pathway such as fructose-1,6-bisphosphate aldolase (Fba1p), glyceraldehydes-3-phosphate dehydrogenase (Thdp), phosphyglycerate mutase (Gmp1p), enolase (Eno1p) and alcohol dehydrogenase (Adh1p).
Conclusion: Taken together, our data indicate that the higher antifungal resistance of the C. glabrata biofilm mode is mediated by stress response pathways. (Supported by Hong Kong RGC Grant #. HKU7624/06M).